Back to the womb

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This month we’re taking a trip back to the womb - and before - to find out about early development. Plus, the importance of placentas, why the age of your womb - rather than your eggs - matters, and a video game-inspired gene of the month.

In this episode

Special stem cells with Peter Rugg-Gunn, Babraham Institute

This month on Naked Genetics we journey back in time to the earliest moments in life as a foetus grows in the womb. Our story starts at the moment of fertilisation, when egg and sperm meet, creating a fertilised egg or zygote...

This single cell, somehow containing all the information needed to make a baby, divides to form an embryo, made up of special stem cells. In turn, these stem cells divide and specialise, eventually making all the tissues of the body. So how does that work?

In search of these hidden biological secrets Kat Arney went on a journey of her own - up the M11 to the Babraham Institute just outside Cambridge - to meet some of the UK’s leading developmental biologists.

Peter Rugg-Gunn, group leader at the Babraham Institute is fascinated by so-called pluripotent stem cells - cells that can turn into all kinds of different tissues. Kat wanted to know why exactly are they so interesting?

Peter – So pluripotency is a particularly remarkable state. Basically, what we can do is we can maintain stem cells in an unspecialised form in the Petri dish and they can undergo this process called self-renewal where they just can continually make new copies of themselves.

But then you can trigger the cells to specialise, to differentiate. You can coax them down different lineages and pluripotency is the ability to then specialise into all of the different cell types in our bodies.

That partly underlies a lot of the excitement and the promise of using stem cells in regenerative medicine - if you want to make a new pancreatic beta cell for instance then pluripotent cells offer a good starting cell material for that.

Kat - When people think about stem cells, they might think about like the stem cells in the very, very early embryo. The little clump of stem cells that become everything in the body and those are often described as totipotent – they can become everything. What's the difference between totipotency and pluripotency?

Peter - Absolutely. So totipotency is the ability of the cells to form any cell type possible so in particular, it includes all the extraembryonic cell types such as the placenta. Pluripotent cells have the ability to form the cells in the adult body. But it can't form cells like the placenta anymore.

I think you’ve touched on a good point there. What's remarkable is that in the human embryo for instance, there's about 10 or so of these pluripotent cells in this 7-day old embryo and these 10 cells make all of the different trillions of cells in our bodies, and I think that’s a very remarkable process.

Kat - It is. That’s why I wanted to become a development biologist. I'm like, “wow! How does that work?” So, how does that work? What is going on when a cell is pluripotent, when it can either make more of itself or go off down a pathway to become other types of cells? What's going on there?

Peter - So I think the important thing to remember here is in the embryo itself, the cells are pluripotent in that they can make all the different cell types, but they don’t remain in a pluripotent or unspecialised state for very long in the embryo. It’s actually quite a transient stage.

In the laboratory, when we grow the human pluripotent cells in a Petri dish, then we can really capture the cells for a long period of time – several years at least – in this undifferentiated state and then drive them to specialise. And so really, it offers a good system in which to ask questions along the lines of what you’ve just asked me.

You know, the simple answer is we don’t know, but we get a lot of cues from different species in development so we can get a good understanding of what signals might be involved from reading about the fruit fly or the mouse, etc. And often, these mechanisms are conserved. And so, we can apply these different chemicals to the human stem cells in a dish and often, they can trigger the same form of cell differentiation.

I think what's exciting in the field right now is we’re also understanding more about how the human embryo develops. There's a lot of discussion and dialogue at the minute about this. Just recently for example, a paper was published where they began to look at the gene expression road map of human pancreatic cells as they develop in the human embryo.

And now, we have this benchmark in which to compare our stem cell differentiation too, so we know whether the stem cells are going down the right line towards becoming a pancreatic cell and how similar is that to how a human embryo does it.

Kat - We’ve talked a lot about embryonic cells and one of the big buzz words at the moment is IPSCs or Induced Pluripotent Stem Cells. So, how do they relate to the kind of pluripotent cells that you find in an embryo? Where do they come from?

Peter - Right. So IPS cells are reprogramed cells, basically. So, this discovery was awarded the Nobel Prize just a few years ago and what the discovery was that you can take any cell type, typically a skin cell, and you can add fairly small set of genes, genetic material which we know is involved in pluripotency.

And over a period of a few weeks, those skin cells slowly transform and reprogram into a cell type that’s very, very similar to a pluripotent cell that you might get from an embryo for example. And they seem to have similar properties. You can grow them in similar ways and then they can re-specialise into any other cell type.

Kat - So now we can take pluripotent stem cells that you’ve got in the lab and you can treat them with certain factors and they will become all sorts of different types of tissues. You can then take cells that have differentiated into a tissue and wind the clock back. This is a very powerful position to be in. So, what can we do with this knowledge? What do we do now that we can wind cells forward and wind them back?

Peter - That’s a great question. One of the things I'm most excited about is, what are the epigenetic and the genetic regulators of this reprograming and then also the forward programing? So, for example, now we can use some of these new genetic tools like CRISPR that’s available to us.

We can then use these technologies to then delete genes at will and then we can ask whether those cells can now reprogram back to a pluripotent state or not. If they can't, that tells us those genes are necessary for reprogramming and then it gives us a clue as to look into those genes and ask why might they be necessary.

So this system is really very powerful way now to try and understand what are regulators of these processes – how do cells get from A to B, basically – and now, we can begin to understand what those regulators might be.

Kat - There's a lot of excitement about using stem cells, using IPS cells for tissue engineering. Okay, you want to build pancreas, let’s take some cells, wind them back, turn them into pancreas cells. Where are the limits? What do we still need to be able to do? I mean, can we basically make all the bits of a human yet? Basically, when am I going to get replacement body parts?

Peter - Yeah. Well a few years ago, that might have been science fiction but now, it’s becoming a bit closer. So there are already clinical trials now out there for diabetes and soon there’ll be for the Parkinson's disease and some of this other degenerative diseases.

Some of the problems we still have, some of the data out there suggested that the cells we’re producing are not truly adult in nature but maybe more foetal-like. And so, that might be okay in some situations but on the other hand, foetal cells are known to express different enzymes and different levels of proteins and things like this.

So whether they can really be truly functional in a disease situation, I think is still not known. Also, we still don’t really know about some of the safety aspects of some of these therapies right now and that again is a big area that people are working hard towards.

Kat - So, you’ve got your pluripotent cells in a dish and you're like, “okay, we want to make heart muscle.” What's the first step? Can you just go straight from stem cell to heart muscle? Do you have to kind of take them through a ‘training program’ basically?

Peter - There's different ways to do it. I think the most effective way, the way most people do it is to try and recapitulate their elements to take them through these sort of building blocks that the embryo uses in order to make heart, for example.

So in terms of that, you would first make mesoderm so you might have a sprinkling of BMP and Wnt in there and then once you made that cell type, you go on to make the next cell type, and so on. And you can really think about it in different stages.

But there are some remarkable studies out there where people seem to have gone directly from one cell type to another cell type, often by overexpressing genes which we know are important in a particular cell type and what intermediate stages they go through? I think who knows, really?

Kat - That sounds like an incredible hack, isn’t it? Like, do not pass, go, just go straight to brain cell.

Peter - Yeah, exactly that. What other cell types can we go straight towards and how does it work?

Kat - When you're thinking about things from an epigenetic point of view, we know that as we go through life, we still keep the same genes but stuff changes. Epigenetic marks get put on, get taken off. Does it matter what age you are when you try and make stem cells? How did those marks get retained or lost as people are moving cells through these different stages?

Peter - Absolutely. So, the data that I'm aware of, if you reprogram a skin cell, for example, from an elderly donor back into one of these pluripotent IPS cells, it seems as though most of the hallmarks of ageing is removed, is reversed essentially, and the cells become young, if you will, again so their methylation or epigenetic clock is set back to zero again.

And then as cells then get re-specialised, it seems like the clock starts again, but in a fairly normal way. So, the resultant specialised cell that you get at the end doesn’t seem to be prematurely aged to any different to what you would expect.

Interestingly, if you make a specialised cell directly from say, an elderly skin cell then in that direct route, it seems to retain these hallmarks of the aged cell to begin with. So it seems like if you go through this pluripotent phase then somehow, some of these hallmarks get removed.

Kat - That’s very interesting. As I get older, I'm like, “Ooh! How can I make myself younger?” So you're saying, I’ll basically have to reprogram all my cells to a pluripotent state and start again.

Reprogramming stem cells to make muscle

Induced pluripotent stem cells - or iPS cells for short - are one of the hottest topics in biology right now, and have the potential to transform medicine...

This month, researchers led by Nenad Bursac at Duke University in the US have announced that they’ve managed to grow the first functioning human skeletal muscle from iPS cells, providing an exciting path towards new treatments for muscle wasting diseases.

Publishing in the journal Nature Communications, the scientists are building on their previous work growing functional muscle tissue from stem cells extracted from small samples of fully-grown muscle tissue, transplanting the cells onto a supportive three-dimensional scaffold that allowed them to grow into fully-formed muscle fibres. But the number of these stem cells is limited, and can only be obtained by taking an invasive muscle biopsy.

This time the team started with iPS cells, which can be made from cells obtained less invasively such as skin or blood, and turned them into muscle stem cells with the help of a protein known as Pax7 - a key transcription factor involved in muscle development.

Impressively, the lab-grown muscle fibres could contract and respond to electrical or chemical signals - just like real muscles in the body. And when they were transplanted into mice, the lab grown muscles soon settled down and started to grow a blood supply, surviving for at least three weeks.

There’s still a lot more work to be done to build up this body-building technology - the lab-grown muscles from iPS cells aren’t as strong as ones grown from muscle-derived stem cells, but the scientists are still hopeful that the technique could be used to develop and test treatments for rare but devastating muscle-wasting diseases and maybe one day even provide new muscle tissue for transplantation.

Inside the egg with Courtney Hanna - Babraham Institute

Now it’s time to wind the developmental clock back even further -all the way back to the egg. Courtney Hanna, a postdoc at the Babraham Institute, is investigating the curious characteristics of mammalian egg cells and the special cells, known as oocytes, that they come from...

She’s using mice as a model and focusing particularly on DNA methylation - an epigenetic mark found around the control switches, or regulatory regions, on DNA that tells cells important information about whether certain genes are active or not, forming a kind of programme for how cells should behave.

As Courtney explained to Kat Arney, taking a closer look at the changes, or reprogramming, of these epigenetic marks as oocytes change into eggs and are then fertilised she’s hoping to understand the events that happen at the very earliest moments of life.

Courtney - So the oocyte differentiates of course as an egg, and it looks like an egg, and has the genes to regulate its function. But it also needs to contain all the information for the embryo when it gets fertilised by a sperm. So we’re really interested in how does it possibly set up these two sets of instructions at the same time.

Kat - It’s remembering that it needs to become an egg cell, so from a precursor cell it’s got to become an egg, and then also it has to then go from being an egg to being a baby.

Courtney - Yup! And we know that the very first steps of an embryo, so after the sperm fertilises the egg, the egg reprograms all of the sperm DNA with maternal proteins and the egg is full of maternal transcripts which create proteins for these early processes. But also, we think that epigenetic information is passed as well, which might instruct the use of the genes in the very first cell divisions.

Kat - What do you mean by epigenetic information and how does that kind of fit with genes and the genetic information that this egg cell is carrying?

Courtney - When I'm talking about epigenetics, there are few different layers of regulation. But in general, you can kind of see it as a set of instructions that either dictates whether a region of DNA is open and accessible or closed and tightly packed. So we look at the different layers of epigenetic information and try to infer whether this is involved in gene regulation itself or in setting up the chromatin state in an open or close state which then might be useful in the embryo.

Kat - So you’ve got two processes going on at once. You’ve got to set up the epigenetic marks for the genes that say, “I'm going to become an egg” and then you’ve got to set up the epigenetic marks that say, “Okay, this is what you need to get the process of building a baby started.”

Courtney - Yup, exactly.

Kat - You’ve got to go from an oocyte, a precursor cell, to making an egg. How do you see the methylation patterns and the epigenetic patterns changing during that process?

Courtney - So, that also hasn’t been looked at that extensively, but when oogenesis starts, there is no methylation throughout the genome and we don’t really understand how the oocyte turns on its set of genes that are required for the oocyte. But as oocyte growth progresses and then an oocyte matures and becomes ready to be ovulated, methylation is acquired during that oocyte growth.

It’s been quite interesting because actually, all of the ground-breaking technologies in low cell numbers have been required to look at eggs because we have to hand collect them so you can only get a few hundred at a time. So, we’ve been really trying to be at the forefront of technology development to try and use molecular tools in these low cell numbers.

And interestingly, everything we seem to look at is abnormal or unusual compared to all other cell types. And so, it’s been quite an interesting road.

Kat - You're taking a handful of mouse egg cells, mouse oocytes, and they look weird to you? What do they look like in epigenetic terms? What's kind of going on in there?

Courtney - So they look really unlike any other mammalian cell type. Actually, the DNA methylation is restricted to such a confined compartment of the genome. It actually really resembles more like insects or plants because they have methylation along gene bodies and that’s what we see in the oocyte. So, it’s thought that this might be really ancestral form of where methylation is laid down.

Kat - Wow! So going way, way, way, way back in our evolutionary history, like a billion years or something, just put those marks on the genes and not on any of the control switches or anything like that, that’s like right at the start of life and right at the start of evolution.

Courtney - Yes, exactly. And so, it is quite the question - how does the egg regulates gene expression if it doesn’t have methylation anywhere at the beginning of oogenesis, but then at the end of oogenesis, we really only see these blocks of methylation over the gene bodies and not at any regulatory regions.

Kat - So then what is going on? You’ve got egg, egg meets sperm, they fall in love, they start the process of making a baby. Then what happens to that pattern that you see where you’ve just got the marks over the genes themselves? What happens next?

Courtney - There's been a few studies published this last year looking at that exact question. So when a sperm enters the egg, what it seems like is almost everything and the sperm DNA is reprogramed. The methylation is erased and there's practically no instructions that seem to be retained from sperm DNA.

Kat - Completely wipe that clean. Wipe it all down. It’s like eww! Wipe it all down!

Courtney - So there was probably only a handful of exceptions we can find. But for the most part, it seems that that’s the case whereas the maternal DNA seems to keep the modifications a bit longer. It’s slower to erase the marks and some marks don’t get erased at all.

But we’ve only been able to look at 3 so far in the community, that’s all that’s been published. And so, we only have sort of the tip of the iceberg of really what is being transmitted.

Kat - What can you do with this kind of information? What does this start to tell us about how development works?

Courtney - I suppose it can really reveal two things. Development is a really unique time where we have a lot of dynamics in epigenetics. So it’s extremely valuable for understanding how the instructions are laid down and how we use certain components of the genome.

In adult tissues, the epigenetic profile is fairly static. And so, we really don’t get the changes and the programming that helps us figure out what instructs what. And then the other aspect is understanding how we get different lineages.

So we start from one cell that contains the instructions for every single cell of the human body or the mouse, or whatever organism you're studying. But we don’t know how at certain points in development, these now split into two groups and this one will make the placenta and these group of cells will make the embryo.

And so, I think we know that epigenetics is key to it, but we really don’t understand how in these group of cells, it would be regulated one way and then these group of cells another.

Kat - Given that these egg cells seem to be managing to control their gene regulation without the kind of patterns of methylations that we might expect then why do we need them in normal cells? What's going on?

Courtney - Yeah. There has been some discussion of the idea of, during these stages of development where we need very dramatic reprograming and reorganisation of the genome to facilitate the transition from an oocyte to an embryo, that maybe transcription has become uncoupled from epigenetics.

And so, it’s an interesting idea to think about maybe some of the regulators that have linked these two processes are downregulated so that we can change the epigenome as much as the cell needs without affecting transcription. And this could be the case.

So when we looked in growing oocytes through the different stages of oogenesis, we actually find there are very few transcriptional changes. So it’s possible they setup the programme early when the processes are still coupled and then by uncoupling the process then the epigenome can regulate entirely independently of gene expression.

Kat - So you just remember what genes you need to switch on to go from oocyte to egg. Just keep doing that and then everything else can kind of go to hell around you.

In praise of the placenta with Myriam Hemberger, Babraham Institute

The stem cells in an early embryo decide whether they’re going to form the embryo itself, or play a supporting role in the placenta and other extra-embryonic tissues. Most developmental biologists focus on the embryo as it grows into a foetus - after all, that’s what becomes a baby once it’s born, so that’s super-interesting - right?

But most cases of pregnancy loss and pre-term birth are caused by problems with the placenta rather than the developing fetus. Yet, as Kat Arney discovered when she talked to Myriam Hemberger at the Babraham Institute, this vital organ has been tragically ignored.

Myriam - Placenta is still often discarded as useless and people don’t really appreciate the importance of the placenta on embryonic development.

So, we were involved in this big phenotyping screen where we had many mouse mutants that were only selected for phenotype that the embryos didn’t survive throughout pregnancy. We looked at the placentas of all of these mutant mouse lines and find that there is 70% of these lines that have placental defects.

What we don’t know is whether the placental defect causes the embryonic lethality but there's a good chance that at least in some cases it will, so it’s still a very overlooked area and that’s what we’re interested in.

Kat - So everyone has just been focusing on the embryo, the baby, because that’s going to be the animal and this wonderful structure that feeds it, that nurtures it, that grows with it just gets chucked in the bin.

Myriam - That’s precisely correct - at least often. Not always, but very often. Too often, I would say!

Kat - So tell me a bit about the placenta. What is it like? What is in there? Because I think I've just had this idea that it is just like a blob of blood and tubes.

Myriam - It does appear like a blob of tubes and blood but this is I guess its function and reflecting its function because you really have the placenta flooded with maternal blood. But then the nutrients and the gases are taken up through those placental so-called trophoblast cells and then go into the foetal circulation, and that is ultimately what feeds the baby, and what makes the baby grow, survive, develop properly.

So if you have a placenta that is insufficient, you have for example babies that are born too small for their age or are just even throughout the entire pregnancy trajectory too small or in the worse cases, won't make it at all and will die during pregnancy. And that’s the same in mice as well as in humans.

Kat - When you started studying these mice that couldn’t make placentas properly, what did you start to find?

Myriam - We started our study by really selecting which embryos don’t make it throughout their gestational period and then went backwards and looked at their placentas to try to establish at least principle cause-effect relationships.

And genetically speaking, genes weren’t selected for anything other than they cause embryonic lethality. So, it was actually a very unbiased approach which is nice because we can now go back to this list of genes and we have over a hundred that we screen very carefully.

This is something we’re doing now - we really go through the genes that we have established to cause a placental deficiency and try to see which generic and general pathways impact on placental development through these factors.

Kat - You're saying that you found a lot of mutations that meant that the embryos died, they didn’t develop properly. Had those genetic mutations formally been thought to be a problem with the embryo and you're saying, “Actually, hang on – you just chucked away the placenta. This is actually a problem with the placenta, not with the development of the foetus.”

Because as a developmental biologist, so many genes, you think, they're embryonic lethal, they lead to no development of the embryo. But these are actually placental problems.

Myriam - That is exactly right and while we cannot obviously prove this in each and every case of such a big screen, this would be correct for – I would think – quite a significant number of instances.

Even historically, there have been a handful of genes where this has been shown. So for example, very prominent exams are the c-Myc proto-oncogene as well as the RB tumour-suppressor, and they have been published already some 20 years ago as causing very specific embryonic phenotypes and defect in the brain and in the heart.

And then some years later, somebody made a genetic rescue experiment where you can, in mice, make the placenta right but the embryo still carries a mutation. And miraculously, the embryos were almost fine so that those were really their first highlight examples that actually that placenta can directly influence embryonic development. And we find that it can specifically influence heart and brain development profoundly.

We don’t know how precisely whether this is through the establishment of blood flow or through shared gene regulatory networks, but it’s definitely the case that in at least, some instances, you will be able to rescue those profound embryonic defects solely with a normal placenta.

Kat - So, if you're thinking about some of the problems that humans have with forming placentas and the number of pregnancies that are lost because of a failed placenta, potentially could this be a way to rescue pregnancies when something is going wrong?

Myriam - That is obviously a long shot and we can't really say that at the moment. But there is that prospect yes, because the benefit of the system so to speak is that the placental cells are only there during pregnancy. So you can envisage that you can manipulate them without impacting the genetic constitution of the embryo itself.

So as long as you can make the trophoblast cells which are these cells that make the placenta functional, that’s all we need. So yeah, there is that long shot prospect but we can't really address that question right now.

Kat - It must seem a bit frustrating that so many of these problems are to do with the placenta, but everyone has just been focused on the embryo.

Myriam - That is at times frustrating. At the same time, it’s a great area for us to be in. So there’s lots of work to be done. People can flood into the field because literally, so much work still to be done from the work I just described with this screen of mouse mutants that has really revealed how little we know about the genetic requirements of placentation. So yeah, we have to learn a lot still on that.

Kat - As well as the mouse mutants that you're working through to find out how these genes affect the growth of their placenta, what other aspects are you looking at?

Myriam - So, in other study that we have just finished is a study where we looked at pregnancy in older females and again, we use the mouse as a model to recapitulate some of the same problems that older women have. They also become more frequent and the woman is more at risk of them as she gets older. So usually, what we call in science ‘advanced maternal age’ unfortunately starts at 35.

Kat – An old crone in your mid-30s!

Myriam - Exactly. So women over 35 but certainly, over 40 do have a higher risk of a whole spectrum of pregnancy complications that actually have nothing to do with the egg per se.

Kat – Ooh your ovaries. It’s like how old are your ovaries and things?

Myriam - That is correct and I guess the same thing about egg freezing and people are obsessed about freezing their eggs when they're younger, in their early 20s perhaps, or even late 20s, early 30s. But what hasn’t really been looked at is that age also impacts on the rest of the woman unfortunately as we could expect.

What we have shown in mice is that when you take embryos from an old female and transplant them into a young female, they actually develop just fine. So, all of these developmental problems can be rescued at least in mice through the context of the younger mother and not to do with the younger oocyte.

What we traced this back to is actually that the uterus undergoes changes as female mice age and the uterine cells are not quite as responsive to the pregnancy hormones so they don’t differentiate as quickly and properly as they should, which means that even that embryo comes and wants to implant, they are not quite as ready to support the embryo.

And whether or not this is the same in humans, we don’t know yet. There will be certainly profound differences because the reproductive cycle in mice and humans is quite different. But it really was meant as a study to lead us into molecular pathways, genetic pathways where we can look in women and see whether they're actually affected as well. Because it is the fact that things like pre-eclampsia and pre-term birth do become more frequent in older women, irrespective of chromosomal defects.

Gene of the Month - Zelda

Found in fruit flies - aren’t they all? - Zelda wasn’t actually this gene’s first name. It was originally known as Vielfaeltig, a German word meaning versatile or diverse, coined by researchers in the German lab where it was first discovered in 2006...

They discovered that a faulty version of the gene affected cell division in fly embryos, leading to many different problems ranging from issues with segmentation and muscle development to abnormalities in the nervous system, suggesting that the product of the gene was highly versatile and played many roles in fly development - hence the name.

But in 2008, a team led by Christine Rushlow in New York published a paper in the journal Nature, showing exactly what the protein encoded by the gene did. They named the protein Zinc-finger early Drosophila activator, abbreviated to ZELDA, reflecting its role in switching on many genes early on in fruit fly development.

Because the name Zelda was snappier and easier to pronounce, and better reflected the actual structure and function of the product, it stuck. Oh, and the fact that a PhD student who worked on the study was a huge fan of the video game Princess of Zelda might have had something to do with it too…

We now know Zelda is a master regulator transcription factor, involved in switching on a huge range of genes in the fertilised egg at the very earliest stages of a fruit fly’s life, playing a major role in shaping the form and function of the insect as it grows and develops.

It works antagonistically with another transcription factor called Grainyhead, with both proteins able to stick to the same sequence of DNA - CAGGTAG, if you’re interested - which is found near the start of many crucial developmental genes, and Zelda seems to hold open the DNA so genes can be switched on.